4. Discussion
The current study revealed, for the first time, that 10 μg/mL ox-LDL
causes necroptosis in foam cells derived from VSMCs and that CTRP9
exerts a potent protective effect against ox-LDL-induced necroptosis.
CTRP9 inhibited the formation of cell necrosomes and the subsequent
inflammation in VSMCs pretreated with ox-LDL. Mechanistically, this
effect is attributable to the upregulation of antioxidant enzymes due to
AMPK activation in VSMC-derived foam cells. Importantly, CTRP9
overexpression significantly suppressed RIP3 and promoted the features
of atherosclerotic plaque stability. These results imply that CTRP9
plays a protective role against atherosclerosis by regulating
VSMC-derived foam cell necroptosis.
Ox-LDL acts as a danger-associated molecular pattern (DAMP) to stimulate
the inflammatory activation of macrophages, VSMCs, and other surrounding
cells [15]. In our study, ox-LDL potently induced cell necrosis and
necroptosis in a dose-dependent manner at concentrations as low as 10
μg/mL. During the formation of atherosclerotic plaques, cellular
turnover in the plaques becomes imbalanced as cells engraft lipids to
form foam cells and undergo both apoptotic and nonapoptotic cell death
[16]. Cell necroptosis leads to inefficient cell clearance, thus
contributing to the acellular necrotic core. Cell necroptosis is
characterized by the disruption of cellular membranes, release of
massive intracellular contents, and expansion of the secondary
inflammatory response, which may play a central role in plaque
expansion. Therefore, considering that ox-LDL is abundantly present in
plaques, our study provides a new perspective on the mechanism by which
ox-LDL promotes plaque progression in atherosclerosis.
Necroptosis is closely associated with inflammation. We found that,
consistent with the cellular characteristics of necroptosis, ox-LDL
induced the secretion of proinflammatory cytokines such as IL-1β, IL-6,
IL-18, and MCP-1. In addition, ox-LDL accelerated the expression of the
macrophage marker CD36 and cell adhesion molecules. Interestingly,
ox-LDL also induced the expression of CD47. CD47, known as the
“do-not-eat-me” molecule, affects effective programmed cell removal
and may lead to clonal expansion. Leeper et al. showed that VSMCs can
produce hyperproliferative cells, which promote inflammation and escape
immune surveillance, further leading to plaque expansion [6]. The
pro-atherosclerosis factor TNF-α participates in the expression of CD47,
which is upregulated in plaques [17]. In contrast, knockdown of the
key antiphagocytic molecule CD47 suppressed the clonal expansion of SMCs
in plaques in vivo [6]. CD47 expression in VSMC-derived foam
cells after ox-LDL treatment may provide a clue to the potential
mechanism underlying VSMC-derived foam cell accumulation in plaques and
may represent a translational target for atherosclerosis treatment.
Oxidative stress response may be associated with ox-LDL-induced cell
death [18,19], and a previous study has indicated that ROS
overexpression induces necroptosis [20]. However, whether mtROS
affect ox-LDL-induced necroptosis in VSMC-derived foam cells has not
been reported. In this study, increased cell necroptosis accompanied by
altered mtROS levels and ΔΨm was observed in ox-LDL-treated VSMCs, but
adding a ROS scavenger effectively reduced cell necroptosis. The results
confirmed that oxidative stress was the basic mechanism underlying
ox-LDL-induced cell necroptosis. We also revealed that CTRP9 exerted a
strong antioxidant effect and that CTRP9 pretreatment prevented the
abnormal accumulation of mtROS and inhibited cell necroptosis induced by
ox-LDL. Consistent with our results, Zuo et al. also reported that CTRP9
restored the ΔΨm and suppressed ROS generation [21]. Nevertheless,
inhibition of the AMPK pathway in VSMC-derived foam cells by
pretreatment with Compound C abolished the CTRP9-induced expression of
antioxidant enzymes and downregulation of cell necroptosis-related
proteins. These results indicate that AMPK activation is crucial for the
protective effect of CTRP9 against ox-LDL-induced damage of VSMC-derived
foam cells. Collectively, the findings of this study reveal the vital
role of mtROS in ox-LDL-mediated necroptosis and the mechanism
underlying the protective effect of CTRP9 against atherosclerosis.
RIP3 deficiency exerts athero-protective effects by reducing the lesion
areas and plaque vulnerability [3,22]. Similarly, RIP1 inhibition
via Nec-1 treatment reverses plaque maturation in
Apoe−/− mice [4]. Knockdown of the necroptosis
executioner MLKL decreases both programmed cell death and the necrotic
core in plaques; however, the total lesion area remaines unchanged
[16], suggesting that MLKL is associated with lipid accumulation in
macrophages. In our study, we found that CTRP9 overexpression suppressed
RIP3 expression in plaque and promoted the features of atherosclerotic
plaque stability, suggesting that cell necroptosis is an effective
target for atherosclerosis treatment. However, the therapies need to be
carefully selected. For example, the advantage of RIP3 may be attributed
to its function in regulating cytokine expression in VSMCs through a
cell death-independent mechanism [23]; thus, a target that can
inhibit both cell necroptosis and inflammation factor expression can
improve clinical benefits. CTRP9 has multiple functions, including
exhibition of anti-inflammatory activity, prevention of oxidative
damage, and inhibition of cell necroptosis. Therefore, CTRP9 may be a
potential drug target in atherosclerosis.